U.S. patent number 5,821,060 [Application Number 08/691,614] was granted by the patent office on 1998-10-13 for dna sequencing, mapping, and diagnostic processes using hybridization chips and unlabeled dna.
This patent grant is currently assigned to Atom Sciences, Inc.. Invention is credited to Heinrich F. Arlinghaus, K. Bruce Jacobson.
United States Patent |
5,821,060 |
Arlinghaus , et al. |
October 13, 1998 |
DNA sequencing, mapping, and diagnostic processes using
hybridization chips and unlabeled DNA
Abstract
A process for deoxyribonucleic acid (DNA) sequencing, mapping,
and diagnostics which utilizes the differences between the chemical
composition of DNA and that of peptide nucleic acids (PNAs) to
provide DNA sequencing, mapping, or diagnostics using natural DNA
fragments, rather than using radioisotopes, stable isotopes or
fluorescent substances to label the DNAs. The process includes the
steps of hybridizing PNA segments to complementary DNA segments
which are fixed to a hybridization surface, or hybridizing DNA
segments to complementary PNA segments which are fixed to a
hybridization surface, and using mass spectrometric or non-mass
spectrometric techniques to analyze the extent of hybridization at
each potential hybridization site.
Inventors: |
Arlinghaus; Heinrich F. (Oak
Ridge, TN), Jacobson; K. Bruce (Oak Ridge, TN) |
Assignee: |
Atom Sciences, Inc. (Oak Ridge,
TN)
|
Family
ID: |
24777250 |
Appl.
No.: |
08/691,614 |
Filed: |
August 2, 1996 |
Current U.S.
Class: |
435/6.12;
435/7.1; 436/173; 536/24.3; 250/423P; 250/282; 530/300; 435/287.2;
435/288.7 |
Current CPC
Class: |
C12Q
1/6874 (20130101); C12Q 1/6872 (20130101); C12Q
1/6872 (20130101); C12Q 2565/501 (20130101); C12Q
2525/107 (20130101); C12Q 1/6874 (20130101); C12Q
2525/107 (20130101); C12Q 2565/627 (20130101); B01J
2219/0061 (20130101); Y10T 436/24 (20150115); B01J
2219/00659 (20130101); B01J 2219/00378 (20130101); B01J
2219/00608 (20130101); B01J 2219/00612 (20130101); C40B
60/14 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 001/68 (); G01N 033/48 ();
B01D 059/44 () |
Field of
Search: |
;435/6,7.1,287.2,288.7
;536/24.3 ;530/300 ;436/173 ;250/282,423P |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Peter E. Nielsen, Michael Egholm, Rolf H. Berg, and Ole Buchardt,
"Sequence-Selective Recognition of DNA by Strand Displacement with
a Thymine-Substituted Polyamide", Science 254, pp. 1496-1500, (Dec.
1991). .
Michael Egholm, Ole Buchardt, Leif Christensen, Carsten Behrens,
Susan M. Freier, David A. Driver, Rolf H. Berg, Seog K. Kim, Bengt
Norden & Peter E. Nielsen, "PNA Hybridizes to Complementary
Oligonucleotides Obeying the Watson-Crick Hydrogen-Bonding Rules",
Nature 365, pp. 566-568, (Oct. 1993). .
Wittung et al., Nucleic Acids Research 22(24):5371-5377 1994. .
Wittung et al., Nature 368:561-563 Apr. 1994..
|
Primary Examiner: Jones; W. Gary
Assistant Examiner: Tran; Paul B.
Attorney, Agent or Firm: Pitts & Brittian, P.C.
Claims
We claim:
1. A method for analyzing hybridized nucleic acids, said method
comprising the steps of:
attaching PNA segments of known sequence to a hybridization
surface;
causing DNA fragments to hybridize to said PNA segments fixed on
said hybridization surface;
rinsing non-hybridized DNA fragments from said hybridization
surface; and
detecting the hybridized DNA fragments on said hybridization
surface by detection of elements or compounds which are exclusive
to DNA as compared to PNA.
2. The method of claim 1 wherein said hybridization surface is
composed of a chemical material selected from the group consisting
of glass, Pyrex.RTM., silicon, silicon oxide, nylon membranes,
polypropylene, gold surfaces, and platinum surfaces.
3. The method of claim 2 wherein said PNA segments attached to said
hybridization surface contain sequences that distinguish normal
from abnormal DNA sequences in a subject gene, thereby enabling
detection of mutations in DNA.
4. The method of claim 1 wherein said hybridized DNA fragments on
said hybridization surface are detected by mass spectrometry
techniques selected from the group consisting of time-of-flight,
quadrupole, magnetic sector and ion trap mass spectrometry.
5. The method of claim 1 further comprising the step of analyzing
said hybridized DNA fragments detected on said hybridization
surface using a surface analysis technique, said surface analysis
technique including at least the step of vaporizing said hybridized
DNA fragments.
6. The method claim 5 wherein said surface analysis technique
further includes the step of ionizing said vaporized, hybridized
DNA fragments.
7. The method of claim 6 wherein said surface analysis technique
steps of vaporizing and ionizing said hybridized DNA fragments are
accomplished simultaneously.
8. The method of claim 7 wherein said surface analysis technique is
selected from the group consisting of secondary ion mass
spectroscopy (SIMS), laser ionization mass spectroscopy (LIMS), and
laser microprobe mass analysis (LAMMA).
9. The method of claim 6 wherein said step of ionizing said
vaporized, hybridized DNA fragments is accomplished using a
resonance ionization method.
10. The method of claim 9 wherein said step of vaporizing said
hybridized DNA fragments which are analyzed using a resonance
ionization method is performed by a technique selected from the
group consisting of ion beam sputtering, sputter-initiated
resonance ionization spectroscopy (SIIUS) using at least one
resonance laser, laser ablation, laser desorption, laser
atomization resonance ionization spectroscopy (LARIS) using at
least one resonance laser, and a thermal technique.
11. The method of claim 10 wherein said at least one laser is
selected from the group consisting of a wavelength tunable laser
and a fixed-wavelength laser that has a coincidental overlap
between their wavelength and an electronic transition in the
element to be analyzed.
12. The method of claim 11 wherein said wavelength tunable laser is
selected from the group consisting of a diode laser, a dye laser,
an optical parametric oscillator, and a solid-state laser.
13. The method of claim 11 wherein said fixed-wavelength laser is
selected from the group consisting of a gas laser and a solid-state
laser.
14. The method of claim 6 wherein said step of ionizing said
vaporized, hybridized DNA fragments is accomplished using a
nonresonance ionization method.
15. The method of claim 14 wherein said step of vaporizing said
hybridized DNA fragments which are analyzed using a nonresonant
ionization method is performed by a technique selected from the
group consisting of ion beam sputtering, laser ablation,
laser-induced desorption, and a thermal technique.
16. The method of claim 15 wherein said nonresonant ionization is
performed using a method selected from the group consisting of a
continuous wave laser, a pulsed laser, electron collision, and
plasma.
17. The method of claim 16 wherein said pulsed laser is selected
from the group consisting of an excimer, a Nd:YAG (Neodinium:
Yttrium Aluminum Garnet), a Cu-vapor, and a sub-nanosecond
laser.
18. The method of claim 5 wherein said surface analysis technique
further includes the step of analyzing said vaporized, hybridized
DNA fragments by a technique which does not require ionization.
19. The method of claim 18 wherein said surface analysis technique
is accomplished using at least one nonionization technique selected
from the group consisting of resonant and nonresonant Raman
spectroscopy, resonant fluorescence spectroscopy, nonresonant
fluorescence spectroscopy, absorption spectroscopy, optical
emission of laser ablated material, and optical emission of ion
beam sputtered material.
20. The method of claim 1 further comprising the step of analyzing
said hybridized DNA fragments detected on said hybridization
surface using a surface analysis technique which does not require
vaporization.
21. The method of claim 20 wherein said surface analysis technique
which does not require vaporization is selected from the group
consisting of Raman spectroscopy, surface-enhanced Raman
spectroscopy (SERS), second-harmonic generation (SHG), Auger
electron spectroscopy, x-ray photon spectroscopy, ellipsometry, and
fluorescence spectroscopy.
22. A method for analyzing hybridized nucleic acids, said method
comprising the steps of:
attaching DNA fragments to a hybridization surface;
causing PNA segments of known sequence to hybridize to said DNA
fragments fixed on said hybridization surface;
rinsing nonhybridized PNA segments from said hybridization surface;
and
detecting hybridized PNA segments on said hybridization surface by
detection of a reduction in surface concentration of elements or
compounds which are exclusive to DNA as compared to PNA.
23. The method of claim 22 wherein said hybridization surface is
composed of a chemical material selected from the group of chemical
materials consisting of glass, Pyrex.RTM., silicon oxide, nylon
membranes, polypropylene, gold surfaces, and platinum surfaces.
24. The method of claim 23 wherein said PNA segments attached to
said hybridization surface contain sequences that distinguish
normal from abnormal DNA sequences in a subject gene, thereby
enabling detection of mutations in DNA.
25. The method of claim 22 wherein said hybridized PNA segments on
said hybridization surface are detected by mass spectrometry
techniques selected from the group consisting of time-of-flight,
quadrupole, magnetic sector and ion trap mass spectrometry.
26. The method of claim 22 further comprising the step of analyzing
said labeled hybridized DNA fragments detected on said
hybridization surface using a surface analysis technique, said
surface analysis technique including at least the step of
vaporizing said hybridized DNA fragments.
27. The method of claim 26 wherein said surface analysis technique
further includes the step of ionizing said vaporized, hybridized
DNA fragments.
28. The method of claim 27 wherein said surface analysis technique
steps of vaporizing and ionizing said hybridized DNA fragments are
accomplished simultaneously.
29. The method of claim 28 wherein said surface analysis technique
is selected from the group consisting of secondary ion mass
spectroscopy (SIMS), laser ionization mass spectroscopy (LIMS), and
laser microprobe mass analysis (LAMMA).
30. The method of claim 27 wherein said step of ionizing said
vaporized, hybridized DNA fragments is accomplished using a
resonance ionization method.
31. The method of claim 30 wherein said step of vaporizing said
hybridized DNA fragments which are analyzed using a resonance
ionization method is performed by a technique selected from the
group consisting of ion beam sputtering, sputter-initiated
resonance ionization spectroscopy (SIRIS) using at least one
resonance laser, laser ablation, laser desorption, laser
atomization resonance ionization spectroscopy (LARIS) using at
least one resonance laser, and a thermal technique.
32. The method of claim 31 wherein said at least one laser is
selected from the group consisting of a wavelength tunable laser or
a fixed-wavelength laser that have a coincidental overlap between
their wavelength and an electronic transition in the element to be
analyzed.
33. The method of claim 32 wherein said wavelength tunable laser is
selected from the group consisting of a diode laser, a dye laser,
an optical parametric oscillator, and a solid-state laser.
34. The method of claim 32 wherein said fixed-wavelength laser is
selected from the group consisting of a gas laser and a solid-state
laser.
35. The method of claim 27 wherein said step of ionizing said
vaporized, hybridized DNA fragments is accomplished using a
nonresonance photoionization method.
36. The method of claim 35 wherein said step of vaporizing said
hybridized DNA fragments which are analyzed using a nonresonant
ionization method is performed by a technique selected from the
group consisting of ion beam sputtering, laser ablation,
laser-induced desorption, and a thermal technique.
37. The method of claim 36 wherein said nonresonant ionization is
performed using a method selected from the group consisting of a
continuous wave laser, a pulsed laser, electron collision, and
plasma.
38. The method of claim 37 wherein said pulsed laser is selected
from the group consisting of an excimer, a Nd:YAG
(Neodinium:Yttrium Aluminum Garnet), a Cu-vapor, and a
sub-nanosecond laser.
39. The method of claim 26 wherein said surface analysis technique
further includes the step of analyzing said vaporized, hybridized
DNA fragments by a technique which does not require ionization.
40. The method of claim 39 wherein said surface analysis technique
is accomplished using at least one nonionization technique selected
from the group of techniques consisting of resonant Raman
spectroscopy, nonresonant Raman spectroscopy, resonant fluorescence
spectroscopy, nonresonant fluorescence spectroscopy, absorption
spectroscopy, optical emission of laser ablated material and
optical emission of ion beam sputtered material.
41. The method of claim 22 further comprising the step of analyzing
said hybridized DNA fragments detected on said hybridization
surface by a surface analysis technique which does not require
vaporization.
42. The method of claim 41 wherein said surface analysis technique
which does not require vaporization is selected from the group of
techniques consisting of Raman spectroscopy, surface-enhanced Raman
spectroscopy (SERS), second-harmonic generation (SHG), Auger
electron spectroscopy, x-ray photon spectroscopy, ellipsometry, and
fluorescence spectroscopy.
Description
TECHNICAL FIELD
This invention is related to the fields of deoxyribonucleic acid
(DNA) sequencing, mapping, and diagnostics. More particularly this
invention takes advantage of the differences between the chemical
composition of DNA and that of peptide nucleic acids (PNAs) to
provide DNA sequencing, mapping, or diagnostics using natural DNA
fragments, rather than using radioisotopes, stable isotopes or
fluorescent substances to label the DNAs.
BACKGROUND ART
The nuclei of living cells possess chromosomes which contain the
genetic information necessary for the growth, regeneration and
other functioning of organisms. Instructions concerning such
functioning are contained in the molecules of deoxyribonucleic acid
(DNA). DNA is contained within the chromosome in a form of
complimentary strands commonly thought of as being configured in a
double helix.
Genetic information in DNA is known to be contained within the
sequence of nucleotide bases which are arranged on a linear polymer
of deoxyribose phosphate. The four bases consist of thymine (T),
adenine (A), cytosine (C), and guanine (G). The two strands of the
DNA double helix are joined in accordance with well known base
pairing rules. These rules provide that T joins with A and that C
joins with G. Accordingly, the base sequence along one strand
determines the order of bases along the complementary strand.
Genetic and diagnostic information can be gathered by determining
the sequence of bases in DNA strands. Heretofore, DNA sequencing
has been accomplished by obtaining DNA from a source of interest
and segregating a template DNA fragment. A complementary DNA
fragment is synthesized by binding a primer ODN to the template
fragment. This template fragment with the primer attached is then
introduced into a solution containing deoxyribonucleoside
triphosphates, DNA polymerase, buffer and magnesium ions.
The polymerase chain reaction, or PCR, is a process which utilizes
a heat stable form of the DNA polymerase to extend the primer and
transcribe the DNA template. The combination of template and
primers is employed to limit the size of the DNA produced. This
size can vary from a few dozen to several hundred or several
thousand nucleotides in the DNA product. During the synthesis of
this DNA product, certain labels are incorporated into the primer
and/or the incoming nucleotides so that the product can be
identified. The region of the genomic DNA that is transcribed is
determined by the sequence of the primers employed. One primer is
used in one strand of DNA and another is used for the complementary
strand. The distance between the binding sites of the two primers
determines the length of the DNA product. In one cycle of the
polymerase chain reaction, the primer binds to the template DNA,
the polymerase transcribes the template DNA beginning at the
primer's 3' terminus and DNA products are released from the
template. These events occur at different temperatures. Template
binding occurs between 35.degree.-50.degree. C., polymerase
activity between 60.degree.-75.degree. C., and the release between
85.degree.-97.degree. C. This cycle is repeated until sufficient
DNA product is made to perform an analysis.
Recent advances in molecular biology and the schedule in the Human
Genome Project have spurred the development of new methods for the
labeling and detection of DNA and DNA fragments. Traditionally,
radioisotopes have served as sensitive labels for DNA while, more
recently, fluorescent, chemiluminescent and bioactive reporter
groups have also been utilized. Fluorescent and chemiluminescent
labels function by the emission of light as a result of the
absorption of radiation and chemical reactions, respectively.
Bioactive labels employ substances derived from living tissue.
The reporter group, .sup.32 P or certain fluorescent or
chemiluminescent substances, are usually incorporated in the
primers or the deoxynucleoside triphosphates to label the newly
synthesized DNA fragments. In the normal application, the DNA
fragments or ODNs are allowed to hybridize respectively to a set of
bound ODNs or DNA fragments that are immobilized on a solid
surface. Because hybridization generally involves the formation of
hydrogen bonds between adenines and thymines and between guanines
and cytosines in opposing DNA strands, the stable binding of one
DNA to another through such hydrogen bonds reveals the sequence in
the DNA fragments if the sequences of the immobilized ODNs are
known. This process is sometimes referred to as "sequencing by
hybridization" (SBH). The DNA on the solid surface is sometimes
referred to as an "SBH chip" or a genosensor chip or a
hybridization surface. This version of the SBH process is referred
to as Format II SBH. E. Southern disclosed Format II SBH in
International Application No. PCT/GB89/00460. Affymetrix, Inc. is
very active in developing commercial products to perform Format II
SBH.
Format I SBH is an alternative method where the genomic DNA is
attached to a solid surface, such as a nylon membrane, and the ODNs
of known sequence and containing labels are allowed to hybridize.
R. Drmanac and R. Czerkvenjakov disclosed Format I SBH in U.S. Pat.
No. 5,202,231, and are developing the analyses commercially at
Hyseq Co.
While the above sequencing technique is capable of producing
reliable results if properly applied, certain disadvantages are
inherent in the process. For example, the ability to resolve
adjacent .sup.32 P labels is limited by the length of the beta
particle track produced by the disintegration of .sup.32 P. With
fluorescent labels, the most common labels in use today,
fluorescence from the solid surface of the SBH chip itself
interferes with the detection of the signal from the fluorescent
label on the DNA. It is common to see a signal to noise ratio of
only 3:1 for fluorescent labels.
The peptide nucleic acid (PNA) is a polyamide type of DNA analog
and the monomeric units for adenine, guanine, thymine and cytosine
are available commercially (Perceptive Biosystems). Certain
components of DNA, such as phosphorus, phosphorus oxides, or
deoxyribose derivatives, are not present in PNAs. As disclosed by
P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254,
1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C.
Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B.
Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind
specifically and tightly to complementary DNA strands and are not
degraded by nucleases. In fact, PNA binds more strongly to DNA than
DNA itself does. This is probably because there is no electrostatic
repulsion between the two strands, and also the polyamide backbone
is more flexible. Because of this, PNA/DNA duplexes bind under a
wider range of stringency conditions than DNA/DNA duplexes, making
it easier to perform multiplex hybridization. Smaller probes can be
used than with DNA due to the strong binding. In addition, it is
more likely that single base mismatches can be determined with
PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer
lowers the melting point (T.sub.m) by 8.degree.-20.degree. C., vs.
4.degree.-16.degree. C. for the DNA/DNA 15-mer duplex. Also, the
absence of charge groups in PNA means that hybridization can be
done at low ionic strengths and reduce possible interference by
salt during the analysis.
Accordingly, it is an object of the present invention to provide a
DNA sequencing, mapping, or diagnostic process in which normal,
unlabeled DNA is used, rather than DNA labeled with stable
isotopes, radioactive isotopes, fluorescent groups, or any other
molecular species attached to DNA for the purpose of
identification. This will eliminate the reagents and labor involved
in labeling the DNA and therefore significantly reduce the analysis
costs.
A further object of this invention is the use of PNA fragments of
known sequence to be used in place of DNA fragments (ODNs) in Type
II SBH. In this case, the presence of DNA hybridization at a
particular PNA sequence location would be determined by detecting
unique components of DNA, such as phosphorus, phosphorus oxide, or
deoxyribose derivatives. The efficiency of detection of phosphorus
and/or phosphorus compounds derived from the DNA should enable
detection of 10.sup.-15 to 10.sup.-18 moles of phosphorus or its
compounds and allow detection of 10.sup.10 to 10.sup.12 molecules
of DNA on a given site.
Another object of this invention is the use of PNA fragments of
known sequence in place of ODNs in Type I SBH. For this
application, PNA which is hybridized to the DNA would cover the
unique components of DNA, such as phosphorus or deoxyribose
derivatives and a surface detection method would see an absence of
these materials at the hybridization location(s).
Yet another object of the present invention is to use mass
spectrometric techniques for measurement of unique components of
DNA, such as phosphorus, phosphorus oxides, or deoxyribose
derivatives, that are not components of PNAs.
Still another object of the present invention is to provide surface
detection methods, including but not limited to Raman spectroscopy,
surface-enhanced Raman spectroscopy, second-harmonic generation on
surfaces, polarization techniques such as ellipsometry, and
laser-induced emission, which can readily identify the difference
between DNA and PNA on hybridized surfaces.
Other objects and advantages over the prior art will become
apparent to those skilled in the art upon reading the detailed
descriptions as follows.
DISCLOSURE OF THE INVENTION
In accordance with various features of the present invention, a DNA
sequencing, mapping, and diagnostic process using unlabeled DNA is
provided. In Format II SBH, the DNA fragments are obtained by
fragmentation procedures, and hybridized to a set of PNAs of known
sequences that are fixed on a solid surface. In Format I SBH, the
DNA fragments are localized on the genosensor chip and the PNAs of
known sequence would be hybridized to the DNAs. The sequence in the
unknown DNA is known from the sequences of the test PNAs to which
it hybridized. The occurrence of hybridization-is determined by
detection of the presence (Format II) or absence (Format I) of
unique components of DNA, such as phosphorus, phosphorus oxides, or
deoxyribose derivatives, at the surface of the genosensor chip.
Detection methods include mass spectrometric techniques and
resonance ionization spectroscopy (RIS). RIS can be used to probe a
spatially resolved portion of a surface by combining it with ion
sputtering (Sputter-Initiated Resonance Ionization Spectroscopy,
SIRIS) or laser ablation (Laser Ablation Resonance Ionization
Spectroscopy, LARIS).
Format II SBH requires that the PNA be attached to a genosensor
surface. The surface to which the PNAs are attached is typically
glass, quartz, or nylon membranes. It is also possible to employ a
gold or platinum film over some solid support and attach the PNAs
to that using chemistries developed by Whitesides and disclosed in
C. D. Bain, E. B. Troughton, Y-T Tao, J. Evall, G. M. Whitesides,
and R. G. Nuzzo, J. Am. Chem. Soc. 111, 321 (1989). The attachment
of the PNA can occur after the synthesis of the PNA sequence that
contains a terminal amine residue. Using silicon chemistry, as
disclosed in Z. Guo, R. A. Guilfoyle, A. J. Thiel, R. Wang, L. M.
Smith, Nucleic Acids Research 22, 5456, (1994), linkers can be made
to attach to a SiO.sub.2 surface or glass surface and provide a
reactive site for the terminal amine on the PNA. Alternatively the
PNAs could be synthesized in situ on the surface of the chip as
described by Fodor et al. in S.P.A. Fodor, J. L. Read, M. C.
Pirrung, L. Stryer, A. T. Lu, D. Solas, Science 251, 767 (1991).
FIG. 1 shows the results of SIRIS analysis of a Format II SBH chip
in which the attached PNA was hybridized with DNA. The DNA is
observed, by phosphorous detection, at the location of
complementary hybridization. Phosphorous is not detected at the
location of the non-complementary PNA, thus distinguishing between
complementary and non-complementary sequences.
Format I SBH requires that the DNA be attached to the genosensor
surface. This is done by using methods similar to those currently
used for Format I SBH using labeled ODNs for detection as noted in
R. Drmanac, S. Drmanac, I. Labat, R. Crkvenjakov, A. Vicentic, and
A. Gemmell, Electrophoresis 13, 566-573 (1992).
Hybridization of test DNA to the sequence-specific PNAs on the
surface is carried out in Format II SBH and hybridization of the
sequence-specific PNAs to the DNA on the surface would be performed
for Format I SBH. The sequence of the test DNA is determined by
scanning the genosensor chip with a position sensitive detector to
detect the presence (Format II) or reduction (Format I) of
phosphorus, phosphorus compounds, and/or organic substances unique
to normal DNA at or near the surface of the genosensor chip.
A nonoptimal resonance ionization spectroscopy (RIS) scheme has
been used to detect phosphorus in adenosine-5'-phosphate. In this
analysis, a series of 5 .mu.l samples of different concentrations
of adenosine-5'-phosphate were deposited and dried onto a silicon
surface. Data obtained from this analysis revealed that the
phosphorus signal was strong and that the strength of the signal
was a function of the amount of the sample. Stronger signals are
obtained when the RIS process is optimized or when negative ion
Secondary Ion Mass Spectrometry (SIMS) or Laser Ionization Mass
Spectrometry (LIMS) is utilized.
The sensitivity of these detection schemes is such that as few as
100-10,000 atoms can be detected, depending on the characteristics
of the element. In the case of phosphorus or phosphorus oxide
molecules, the higher detection level would apply, which
corresponds to .about.10.sup.-19 mole, a level that is comparable
to the amounts of radioisotope or fluorescent labels used in other
procedures. The novel aspect of this invention is that no extrinsic
label is required since the phosphorus of the test DNA is used to
detect its presence on the PNA site on the chip. Furthermore, each
base in single stranded DNA contains one phosphorous atom and
therefore, a five hundred base fragment will contain five hundred
phosphorous atoms available for detection.
The main requirements for the detection technique are that it be
very sensitive, have good spatial resolution, and be capable of
distinguishing the unique components of DNA, such as phosphorus or
deoxyribose derivatives. Several mass spectrometric techniques fall
into this category; these include SIRIS, LARIS, secondary ion mass
spectrometer (SIMS), laser ionization mass spectrometry (LIMS, also
known as laser microprobe mass analysis or LAMMA), and nonresonant
or sub-nanosecond post-ionization techniques. Among the non-mass
spectrometric methods that are also suitable are: atomic force
microscopy, reflectance spectroscopy, second harmonic generation on
surfaces, polarization techniques such as ellipsometry, Raman
spectroscopy, and surface enhanced Raman spectroscopy.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A. FIG. 1A shows a diagram of an SBH chip on which two
different PNA oligos that were attached in separate locations.
Position A=PNA oligo which is complementary to the M13(-20) DNA
oligonucleotide probe and position B=PNA oligo which is not
complementary to the M13(-20) DNA oligonucleotide probe. The DNA
chip was hybridized with the M13(-20) DNA oligonucleotide.
FIG. 1B. FIG. 1B displays a SIRIS (sputter-initiated resonance
ionization spectroscopy) image of the phosphorus detected on the
surface of the SBH chip. Phosphorous is observed where the DNA
nucleotide is located, demonstrating successful detection of
hybridization of DNA to PNA.
BEST MODE FOR CARRYING OUT THE INVENTION
PNA oligomers are synthesized like DNA oligomers in that the
synthesis begins with the four different monomers and links them
together to form the oligomer of desired sequence. In the case of
PNA, the monomers for making PNA each contain one of the four bases
(adenine, guanine, thymine, or cytosine) attached to 2-aminoethyl
glycine. PNA monomers have amino and carboxyl termini, which are
similar to amino acids. PNA monomers are linked by peptide bonds to
form an oligomer and the synthesis protocols required to link the
monomers are the same as those used for standard peptide synthesis.
Therefore, the PNA oligomers can be synthesized on a resin and then
cleaved from that resin and applied to the SBH surface.
Alternatively, they can be synthesized directly on the SBH chip as
is currently being done with DNA oligomers, using photolithography,
physical masking, or ink jet application techniques.
Using the polymerase chain reaction (PCR), defined strands of
double-stranded DNA can be replicated for hybridization to the set
of known-sequence PNAs in the SBH Format II process. After
hybridization of the DNA to the PNA, DNA will be attached only at
sites containing complementary PNA sequences. The genosensor chip
can be inserted into a vacuum chamber for Laser Ionization Mass
Spectrometry (LIMS) analysis of the PNA sites. In the LIMS
analysis, a laser is used to desorb a small amount of material from
the PNA sites. A small fraction of this material will be ionized
and the resulting ions can be mass analyzed by a mass spectrometer.
In particular, the negative phosphates ions and negative ions of
other phosphorus compounds will indicate that DNA is present and
therefore hybridization has occurred.
LIMS may be employed to scan a SBH chip mounted on a support by
moving the support while the laser beam position remains fixed.
Alternatively, the sample can remain fixed in position and the
surface scanned rapidly with the laser beam. The laser beam can be
focused to <10 .mu.m in diameter to improve the resolution
between adjacent DNA positions of the chip. It is also possible to
use a large spot size laser beam to simultaneously desorb sample
from many or all of the hybridization positions on the chip, and
image the resulting ions on the detector.
Those skilled in the art will recognize that the signal from
surface analysis techniques such as LIMS or other surface analysis
techniques such as those methods described below will indicate the
reduction in surface concentration of constituents of the target
material (for example, DNA) when it is covered by a different
material (for example, PNA) that does not contain those
constituents. In this way, the above technique is adaptable for use
in Format I SBH where DNA is attached to a surface and PNA is
hybridized to the immobilized DNA.
Those skilled in the art will also recognize that the small number
of ions produced in the LIMS process can be enhanced by nonresonant
laser ionization (surface analysis by laser ionization, SALI) or
resonance laser ionization (LARIS).
Those skilled in the art will further recognize that the ablation
laser in LIMS can be replaced by an ion beam, as in SIMS, sputtered
neutral mass spectrometry (SNMS), or SIRIS.
Moreover, those skilled in the art will recognize that several
non-mass spectrometry techniques, such as second harmonic
generation on surfaces, Raman, surface enhanced Raman,
ellipsometry, and reflectance spectrometry can also be used to
monitor the surface composition.
Those skilled in the art will readily recognize that the present
invention could also be practiced for determining RNA sequences.
One skilled in the art could obtain DNA by reverse transcription of
RNA from the source of interest.
Data from the LIMS analysis can be displayed digitally or in
graphic form that is comparable to images obtained from
autoradiography or fluorescent analyses. The sequence obtained
could be printed out directly and any uncertainties designated.
Relative to radiation safety, the purchase and disposal of
radioisotopes is eliminated and the process is safer for
personnel.
From the foregoing description, it will be recognized by those
skilled in the art that the present DNA sequencing, mapping, and
diagnostic processes, which utilizes hybridization and unlabeled
DNA, offer several distinct advantages over the prior art.
Specifically, the DNA Sequencing, Mapping, and Diagnostic Processes
Using Hybridization Chips and Unlabeled DNA provides costs savings
by eliminating the time and chemicals needed by other techniques to
label the DNA or substance hybridized to the DNA (ODNs or
PNAs).
While a preferred embodiment has been shown and described, it will
be understood that it is not intended to limit the disclosure, but
rather it is intended to cover all modifications and alternate
methods falling within the spirit and the scope of the invention as
defined in the appended claims. Having thus described the
aforementioned invention,
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